* Part B reverse primer: 5'-GAAGCCTGCAGCGGCCGCTACTAGTA-3' (reverse complement of Part A forward primer)

* Part B reverse primer: 5'-GAAGCCTGCAGCGGCCGCTACTAGTA-3' (reverse complement of Part A forward primer)

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2. Perform a PCR reaction with a high fidelity polymerase such as Phusion using the recommended protocol. For template DNA, dilute miniprepped BioBrick plasmid DNA 1:1000 to minimize these “background” plasmids from getting transformed later.

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2. Perform a PCR reaction with a high fidelity polymerase such as Phusion using the recommended protocol. For template DNA, dilute miniprepped BioBrick plasmid DNA 1:1000 to minimize these “background” plasmids from getting transformed later. Digestion with DpnI after the PCR reaction may reduce background plasmids, but in my experience there is a high success rate (>50%) without the use of DpnI or phosphatase on the vector.

Revision as of 04:12, 8 April 2009

Contents

Overview

This is a protocol to assemble two BioBricks using the Clontech In-Fusion PCR Cloning Kit. This protocol allows for maintaining BioBrick standard formats and introducing mutations with two parts in a single step. One PCR-amplified BioBrick has homology on each end with the second PCR-amplified BioBrick (vector amplified with the BioBrick) to allow for the fragments to be fused together in the In-Fusion reaction. This procedure can be adapted to fuse more than two fragments together or to re-engineer existing BioBricks. The advantages of In-Fusion BioBrick assembly over standard assembly are that it is faster, does not require restriction digestions or ligations, and is more flexible in the sense that there is more control over the exact engineered sequence. The disadvantages are that it is more expensive, custom primers are required, and occasionally there are mutations in assembled plasmids. However, sequencing a few miniprepped plasmids usually results in at least one plasmid without mutations.

Materials

Thermocycler

Primers

Phusion (or another high fidelity) PCR Mastermix

Qiagen PCR Purification Kit

Nanodrop (not required, but very useful)

In-Fusion PCR Cloning Kit

Gel box, power supply, and gel supplies

Qiagen Miniprep Kit

Procedure

1. Order primers of parts to assemble.

For Part A (upstream part) + Part B (downstream part) with the pSB1A2 vector (this protocol can be adapted for different vectors by changing the sequences below)

Part A Forward primer: 5'-TTCTGGAATTCGCGGCCGCTTCTAG-3' (specific to the pSB1A2 prefix + 5 bases upstream of the prefix)

Part A Reverse primer: last 20 bases Part A + scar (if wanted) + first 20 bases Part B (reverse complement)

Part B + vector Forward primer: reverse complement of Part A reverse primer

Part A + vector Forward primer: 5'-TACTAGTAGCGGCCGCTGCAGGCTTC-3' (specific to the pSB1A2 suffix + 5 bases downstream of the prefix)

Part A + vector Reverse primer: last 20 bases Part A + scar (if wanted) + first 20 bases Part B (reverse complement)

Part B Forward primer: reverse complement of Part A reverse primer

Part B reverse primer: 5'-GAAGCCTGCAGCGGCCGCTACTAGTA-3' (reverse complement of Part A forward primer)

2. Perform a PCR reaction with a high fidelity polymerase such as Phusion using the recommended protocol. For template DNA, dilute miniprepped BioBrick plasmid DNA 1:1000 to minimize these “background” plasmids from getting transformed later. Digestion with DpnI after the PCR reaction may reduce background plasmids, but in my experience there is a high success rate (>50%) without the use of DpnI or phosphatase on the vector.